The experiments to be described in this thesis were undertaken to obtain stocks of the fungus Aspergillus nidulans containing translocations between various pairs of linkage groups. The translocations were identified genetically, in gamma-irradiated diploid colonies. The chromosomes in this fungus are very small, but some can be identified individually (Elliott, 1960). The stocks characterized here will be used later in an attempt to associate each chromosome with the appropriate linkage group. The method used located not only translocations, but recessive lethals and some point mutants as well.

The artificial production of malformations, teratogenesis, was established as a special branch of experimental biology during the nineteenth century. Outstanding among the pioneer workers was Dareste who on the basis of extensive observations on the effects of temperature variations on chick embryos, concluded that teratogeny was always the consequence of a deviation from normal embryonic pathways, and that almost all malformations were caused by arrest of development. He further noted that induced defects in the chick closely resembled those found in mammals, especially in man.

Species of the genus Lotus (Leguminosae) are principally located in two geographic areas: the Pacific coast of North America and the Mediterranean region. In addition, endemic species are found in such widely scattered places as South Africa, Australia and Japan. [...]

Epilepsy, from the earliest times, has been branded 'hereditary’, and marriage and children for those "possessed" by this ailment was frowned upon. To Hippocrates and Galen (115) idiopathic epilepsy was that type which developed in the brain directly and was assumed to have a hereditary basis. Burton (84) in his "Anatomy of Melancholy", claims that the ancient Scots "instantly gelded" any man with falling sickness, and if a woman "were round to be with child, she with her brood were buried alive and this was done for the common good, lest the whole nation should be injured or corrupted".

Spontaneous abortion in man is a subject important to workers in many biological fields -- to the obstetrician who must deal not only with the event itself, but also with its psychological and social aspects; to the demographer who should take account of it in his analyses of population structure and change; to the investigator of reproductive physiology who attempts to find the cause of what is one of the most frequent abnormalities of pregnancy; to the geneticist who tries to assess the role of heredity in the production of such abnormalities; and to the biometrician who is required to find techniques capable of dealing with the statistics of abortion.

Over centuries, crop domestication and improvement has led to modern commercial agriculture. Agricultural biotechnology is considered by many a natural step in the course of crop improvement by utilizing genetic engineering. Currently, the global production of biotech crops is approximately 34% of global agriculture. The major biotech crops in terms of production volumes are canola, cotton, maize and soybean. In Africa, South Africa is the only country to accept and commercially produce genetically modified (GM) crop. The 2007, GM traits per crop with environmental release status in South Africa included insect resistant (IR) and herbicide tolerant (HT) cotton (including the stack for both traits) (90% of total cotton production), IR and HT maize (including the stack for both traits) (57% of total production) and HT soybean (80% of total production). There are several factors that impact on the application of this technology in terms of commercial as well as small scale farming. These include: intellectual property rights, socio-economics, regulatory frameworks, agriculture, environment, niche markets and cost benefit. Of all of these aspects, gene flow from GM to non-GM or organic products, land races and wild relatives is a critical consideration. In this study, the impact of potential pollen mediated gene flow (PPMGF) and pollen mediated gene flow (PMGF) was studied in GM soybean and maize, two of the most important GM food crops in terms of production volumes. In this study, GM gene flow was found to have occurred up to 0.9 m from a GM source at two locations over two seasons, despite being considered a selfpollinating crop (Greytown 2005/2006 and Delmas 2006/2007, respectively). However, it was also found that GM soybean pollen was not wind borne and we suggest that the gene flow observed was due to insect-mediation. Future studies of PPMGF in South Africa should include a survey of insects present with the potential to act as a pollen vector in soybean. In the maize component of this study, molecular technology was used to detect GM maize pollen up to 400 m from a GM pollen source. Furthermore, it was found that out-crossing of GM to non-GM maize was possible at a distance of 300 m from the GM field. Based on the statistical analysis of out-crossing data, I have determined that the average theoretical zero (0.0001%) level of out-crossing was between 1.3 km and 2.0 km over different geographic locations. However, what was unexpected is the difference in out-crossing per location for a specific direction. For example, in Bainsvlei (2005/2007) for the ENE direction, the calculated distance to achieve 0.01% out-crossing is 79 km, yet the average is 113 m. Similarly in the second season for the same direction, the calculated distance is 956 m and the average is 135 m. The implication of these data is that it is not possible to establish a one size fits all isolation distance to minimize or prevent gene flow. Different threshold levels of commingling require different isolations distances and should be determined by the acceptable level of tolerance for commingling. For non-GM production in South Africa, based on the 1.0% threshold applied by the Department of Agriculture, I suggest a minimum isolation distance of between 120 m up to 200 m, assuming that the weather patterns are comparable to those of the current study as well as that the non-GM seed being planted contains 0% GM. However, for more stringent thresholds, the isolation distance would need to be extended. For organic crop production, at 0% adventitious GM, as well as field trials of second and third generation GMOs, it is suggested that the isolation distance be set at a minimum of 1.5 km and 2.0 km, respectively. In addition, for non-GM seed production (with a mandatory 0% tolerance so as not to contravene patents) I recommend a 1.5 km isolation distance. These suggested isolation distances are based on the absence of time isolation. It is hoped that this study will help to inform regulatory as well as on farm decision making and that it could be used as a blueprint for other GM crops, especially indigenous African crops such as sorghum and cassava.

The genus Clivia Lindl., which belongs to the family Amaryllidaceae J. St-Hil. (1805), is comprised out of seven different species. Clivia nobilis, C. caulescens, C. miniata, C. gardenii, C. mirabilis, C. robusta and the natural hybrid Clivia xnimbicola all forms part of this genus. Clivia mirabilis is found in the Northern Cape Province and is geographically isolated from the six other species which grows along the Eastern Coast and escarpment of South Africa. Conrad et al. (2003) proved that C. mirabilis and C. nobilis were the two most primitive species in the genus Clivia. During this study sequencing results were used to detect barcodes/SNPs for C. nobilis and C. mirabilis and to reveal genetic variation between the Clivia species. Clivia nobilis and C. mirabilis were tested with cross-species microsatellite makers to reveal intraspecific variation. Seven different gene regions were sequenced. Six were chloroplast regions, namely the atpH-I, matK, rpoB, rpoC1, rpl16, the trnL-F regions and one was a nuclear region, ITS1. The regions used for sequencing were evaluated as potential barcoding/SNP regions for future use. They were also used to infer the evolutionary development of C. nobilis and C. mirabilis. All seven Clivia species were analysed but this study focused mainly on morphologically different specimens of C. nobilis and C. mirabilis. The sequences were aligned and edited with Geneious Pro. A total of forty-seven polymorphic sites were observed between al seven species. Within the rpl16 region eleven parsimony informative sites were observed. The matK and trnL-F regions each had eight parsimony informative sites. ITS1 had three sites and rpoB and rpoC1, one parsimony informative site each. Within the atpHI region no parsimony informative sites were observed. The sequencing data obtained could be used for species identification and, therefore, showed great potential as barcoding regions. We propose that matK, rpl16 and trnL-F are used as a barcode in C. nobilis and C. mirabilis because they had the most parsimony informative sites. The cladogram obtained from the combined data set (atpH-I, rpoB, rpoC1, matK and trnL-F) confirmed that C. nobilis and C. mirabilis are two separate species. Clivia caulescens and C. xnimbicola forms a monophyletic group. Within the rpl16 chloroplast region intraspecific variation in C. mirabilis and interspecific variation between C. nobilis and C. mirabilis were observed. The phylogentic tree representing the sequencing results of the rpl16 region revealed three distinctive groups within the four different C. mirabilis populations. Two plants within one of the Donkerhoek populations showed more variation than the rest of the population. The rpl16 gene region proved to be ideal in order to test intraspecific variation in C. nobilis and C. mirabilis. To evaluate the use of cross-species markers, microsatellite makers designed for Phaedranassa tunguraguae, Hymenocallis coronia and Clivia miniata were tested on C. nobilis and C. mirabilis. Although amplification was obtained, in most cases the results could not be optimized in order to provide reliable analysis. In future species specific primers for C. nobilis and C. mirabilis will be developed. This study undoubtedly identified barcodes/SNPs for C. nobilis and C. mirabilis which can be used to eliminated mistaken identity. Gene regions specific for intra- and interspecific variation were identified and can be used in future for population studies.

Anthocyanins belong to a large group of secondary plant metabolites, the flavonoids, and fulfil a range of biological functions that include the cyanic pigmentation they provide to flowers, fruits, vegetables and leaves. The anthocyanin biosynthetic pathway has been well elucidated and much effort has been made by researchers to modify some of the catalytic steps, thereby changing the colour of some ornamental and cut flower species. The genus, Clivia, is an ornamental monocot indigenous to South Africa and there has been a growing interest among local and international Clivia breeders to introduce novel flower colour varieties into the market. Transgene technology holds new possibilities to ensure modification of Clivia flower colour. However, the genetics and biochemistry of the Clivia anthocyanin biosynthetic pathway must first be investigated before any attempts regarding biotechnology can be made. The current study is the first to deal with the identification and expression analysis of flavonoid biosynthetic genes in the genus Clivia, specifically those involved in anthocyanin biosynthesis, thus identifying future prospects and motivating research in unexplored territory. A previous study concerning an HPLC analysis of Clivia anthocyanin content confirmed the presence of cyanidin and pelargonidin derivatives as the main pigments in the tepals and fruits. This enabled the establishment of a putative Clivia anthocyanin biosynthetic pathway illustrating each enzymatic event. Conventional PCR with degenerate primers and a tepal cDNA template was used to isolate four different target sequences. Consensus cDNA fragments of 586 bp, 326 bp, 510 bp and 225 bp confirmed the existence of Clivia orthologues for Chalcone synthase (CHS), Chalcone isomerase (CHI), Flavanone 3-hydroxylase (F3H), and Dihydroflavonol 4-reductase (DFR), respectively. The deduced amino acid sequences of CHS, DFR and F3H harboured important conserved residues that confirmed the existence of functional enzymes. Furthermore, nucleotide sequence analyses between each new Clivia cDNA fragment and the corresponding fragments of other higher plants, regarding similarity/identity and phylogeny demonstrated closer homologies and evolutionary relatedness to other monocot species. The identification of the Clivia flavonoid biosynthetic genes enabled the expression analyses of CHS and DFR. These structural genes encode enzymes responsible for two important controlling steps necessary to determine the nature of the final end-product(s) of the pathway. Real-time quantitative RT-PCR involving SYBR® Green chemistry was used to investigate the temporal expression of the two genes in the tepal, stamen and carpel tissues during five flower developmental stages of an orange and yellow variety of Clivia miniata. Statistical analyses were used to support any findings where possible. Each respective tissue type revealed its own trend in expression for both CHS (an early biosynthetic gene) and DFR (a late biosynthetic gene) throughout flower development except in the stamens of the yellow flowers. These findings suggested the co-ordinate regulation of the Clivia miniata anthocyanin biosynthetic genes as a single module, a model of transcriptional regulation that is often found in certain monocot species (Dooner et al., 1991; Meldgaard, 1992; Martin and Gerats, 1993). To understand the regulatory system that confers flowers colouration, genes that encode transcription factors should be isolated and their spatial and temporal expression investigated. The âparallelismâ between anthocyanin biosynthetic gene expression and anthocyanin production in the tepals of the orange and yellow Clivia miniata varieties was also investigated. UV-visible spectrophotometry at A530nm was used to quantify total anthocyanins at each developmental stage after extraction. At full bloom the orange flowers had almost 16 times more anthocyanins, which support orange colour development, than the yellow flowers. It was confirmed by the outcomes of statistical analyses that the trends in expression of CHS and DFR and anthocyanin production were similar. Methods such as HPLC are recommended for more precise qualitative and quantitative determination of total monomeric anthocyanins.

Genetic management has become a critical part of the overall management of nonhuman primate populations. This dissertation describes a genetic analysis of the chacma baboon population at the Suikerbosrand Nature Reserve. The aim of this study was to apply genetic data as a credible tool to contribute to the conservation and management of chacma baboons at Suikerbosrand Nature Reserve. The specific objectives included individual identification, determining genetic relationships and levels of gene flow within- and among the fourteen troops, and to construct a genetic database with individual genotypes of the whole population. A secondary objective of this study was to determine whether it would be feasible to extract DNA from fecal samples collected from a sleeping site and then use the genetic profiles to determine the number of individuals in that specific troop. The current population is estimated to be between 611 and 764 animals. The sleeping site of the Diepkloof troop was used for this part of the study. A panel of eleven human microsatellite markers was used for DNA analysis. DNA profiles from all the blood samples were successfully constructed and could be used to estimate genetic relationships. The level of genetic diversity in the Suikerbosrand baboon population did not differ significantly from that in the outgroup. Thus, the reintroduction of new individuals into the population to maintain acceptable levels of diversity is not an immediate priority. High levels of gene flow were observed between the troops, especially the troops located in the central part of the reserve. In order to ensure high DNA quality from fecal samples collected at the sleeping site, the collection method for fecal samples were optimized (A manuscript based on the work in this section has been accepted for publication in the European Journal of Wildlife Research). The profiles obtained from the fecal samples that were collected at the Diepkloof site corresponded with two of the thirteen profiles from the reference database. The estimated size of the Diepkloof troop is thirty seven individuals. The results show that non-invasive sampling could be a promising alternative for future research on the reserve, as the samples can be used to determine individual profiles. The genetic data collected can be combined with ecological and behavioral information collected form future research to further understand the population structure of the Suikerbosrand chacma baboons and changes that might occur in the population.

The recent taxonomic review of the legless, fossorial skink genus Acontias by Daniels et al. (2006), using DNA sequence data, led to the erecting of the new genus Microacontias for all the small-bodied taxa. These are Microacontias lineatus lineatus, Microacontias lineatus tristis, Microacontias lineatus grayi and Microacontias litoralis. The latter species was, however, nested in the lineatus complex which raised questions about its true taxonomic status. Furthermore, the taxonomic status of the lineatus complex is questioned following the poor relationships between taxonomic diversity and morphological character states in these sub-terrestrial skinks (Daniels et al., 2002, 2005, and 2006). This study therefore addresses the following two questions: (i) should the taxonomic status of the lineatus complex be accepted as it stands and (ii) is Microacontias litoralis an independent species or does it form part of the lineatus complex. It is hypothesized that M. l. lineatus and M. l. tristis do not represent separate taxa (near identical morphology), while M. l. grayi and M. litoralis should be given independent status. The diagnostic methods utilized were (i) Mitochondrial DNA (mtDNA) sequence data (16S rRNA and Cytochrome b), (ii) morphometric data comprising head and body measurements, and (iii) meristic data comprised of various scale counts. The findings suggest that at least in terms of the gene fragments sequenced, the subspecies of the lineatus complex are very closely related. The phylogeny reports no distinct grouping of the taxa, nor could the morphological analyses separate the taxa as independent evolutionary units. The low genetic divergence but extensive color variation can be viewed as an example of morphological diversification despite genetic conservatism.